The present invention relates generally to immunological analysis and more particularly to a method of analyzing automatically given substances in samples due to an immunological reaction of antigen and antibody.
At present, due to progress in medical treatment, very small amounts of biological substances in samples can be analyzed and this contributes to early diagnosis for various diseases. For instance, malignant tumors such as .alpha.-fetoprotein and carcinoembryonic antigen, diseases in abnormal secretory of hormone such as insulin and thyroxine, and immunological diseases such as immunoglobulin can be diagnosed in early stages and further the monitoring after treatments for these diseases can be carried out reliably. Moreover, the measurement of incomplete antigens, i.e. low molecular hapten of medical substances contributes to development of a plan of medication.
Many biological substances are analyzed in an immunological manner by utilizing the antigen-antibody reaction and various methods for effecting the immunological analysis have been developed. For instance, existence or non-existence of agglinated clots of antigen-antibody compound formed by the antigen-antibody reaction is detected by agglutination method, sedimentry method, nephelometry method, etc. to analyze desired biological substances. However, in the known methods, since the sensitivity is low, a large amount of antigen-antibody compound is required and only qualitative analyses or quasi-quantitative analyses can be performed. In order to avoid such a drawback, there have been further proposed the following methods. In one of the known methods, antigen or antibody is bound with carbon or synthetic resin fine particles which are then subjected to the antigen-antibody reaction with the biological substances to be analyzed and the substances are detected by means of the agglutination method or nephelometry method. In another known method, antigen-antibody compounds are detected at a high sensitivity by using antigen or antibody marked with labeling material such as radioisotope, fluorescent material, luminescent material and enzyme. However, since the former method is inferior to the latter method in the sensitivity, recently the latter method using the high sensitivity labeling substance has been predominantly adopted.
The analytic methods using the markers are classified into radio-immuno-assay using radioisotope tracers, fluorescent-immuno-assay using fluorescent labeling material, and enzyme-immuno-assay using enzyme markers. Among these methods, the enzyme-immuno-assay has been particularly developed owing to the reason that it does not require special installation and measuring technique and can be performed easily by using commonly developed colorimeters. The enzyme-immuno-assay is further classified into homogeneous enzyme-immuno-assay and heterogeneous enzyme-immuno-assay. In the homogeneous analysis, a variation in activity of labeling enzyme due to existence or non-existence of the immunological reaction is directly measured to detect substances to be analyzed. In the heterogeneous analysis, use is made of insoluble carriers such as glass beads or synthetic resin particles on which antigen or antibody has been fixed, enzyme-labeled antigen or antibody bound with the antibody or antigen fixed on the carriers and free enzyme-labeled antigen or antibody not bound with the antibody or antigen on the carriers are separated from each other by washing treatment, and then an activity of labeling enzyme is detected to measure a quantity of substances to be analyzed. Hereinbelow, the process for separating the bound antigen or antibody and the free antigen or antibody from each other is termed as B-F or bound-free separation for the sake of simplicity. Although the homogeneous analysis can be performed by simple processes, it can analyze only the low molecular hapten such as medical substances, but cannot analyze high molecular biological substances. Contrary to this, in the heterogeneous analysis, although the washing process is required for effecting the B-F separation, it can be applied to any kinds of low and high molecular substances. Therefore, recently the heterogeneous enzyme-immuno-assay has been generally adopted.
In the heterogeneous enzyme-immuno-assay, there have been developed competitive method and sandwich method. Now these methods will be explained with reference to the drawings.
FIG. 1 illustrates successive steps of the competitive method. Given antigen or antibody which reacts with antibody or antigen substances 2 of a sample has been previously fixed to an outer surface of a insoluble carrier 1. At first, the antigen-antibody reaction is carried out between the antigen or antibody fixed onto the carrier 1 and the antibody or antigen 2 in the sample as well as a labeled reagent 3 which has been prepared by labeling substances same as the substances 2 to be analyzed with enzyme marker. Then, a washing process is carried out to effect the B-F separation between the substance 2 and labeled reagent 3 bound with the carrier 1 due to the antigen-antibody reaction and free substances 2 and reagent 3 which are not bound with the carrier 1. Next, a color reagent which selectively reacts with the labeling enzyme is added and a reaction liquid is colorimetered to detect the enzyme activity of the labeling enzyme.
FIG. 2 shows successive steps of the sandwich method in which use is made of an insoluble carrier 5 having fixed thereto antibody or antigen which is reactive with antigen or antibody substances in a sample to be tested. At first, the carrier 5 and the sample 6 are mixed to effect the antigen-antibody reaction between the substances 6 in the sample and the antibody or antigen fixed to the carrier 5. Then, the B-F separation is carried out by means of the washing step. Next, a labeled reagent 7 is added to effect the antigen-antibody reaction. The labeled reagent is prepared by marking with enzyme substance selectively reacting with the substance 6 to be analyzed. Then, after the B-F separation is effected again, a color reagent reacting with the labeling enzyme in the labeled reagent 7 is added and a test liquid thus obtained is colorimetered to detect the activity of the labeling enzyme.
As explained above, in the heterogeneous immuno-assay the B-F separation has to be carried out once in the competitive method and twice in the sandwich method during the analysis for respective sample and further if a reaction vessel for effecting the antigen-antibody reaction is used repeatedly, there must be further provided a step for washing the reaction vessel after the end of analysis for a sample, but before the start of analysis for another sample. In case of automating the enzyme-immuno-assay requiring at least two washing steps including the B-F separation, separate washing devices may be provided at different positions. However, then an automatic analyzer is liable to be large in size, complex in construction and expensive in cost. This disadvantage will also appear in automatic analyzer effecting radio-immuno-assay and fluorescent-immuno-assay.
In a Japanese Patent Application Laid-open Publication No. 74,662/82 published on May 10, 1982, there is disclosed an automatic enzyme-immuno assay apparatus in which the B-F separation is effected by removing a carrier having antigen-antibody compound bound thereto from a reaction vessel to another reaction vessel. It is apparent such an analyzer becomes large in size and requires a special mechanism for transporting the carrier. Further, the efficiency of analysis is low and a number of samples could not be processed promptly. In a Japanese Patent Application Laid-open Publication No. 124,254/82 published on Aug. 3, 1982, there is described an automatic analyzer for effecting an immunological analysis in which the B-F separation is performed by rotating a rotor on which reaction vessels having carriers contained therein are arranged. In this analyzer, since the excess liquid is discharged in all directions due to the centrifugal force, the treatment of the discharged liquid becomes cumbersome. Further, since the reaction vessels containing the carriers are repeatedly used, a special treatment for releasing the antigen or antibody from the carriers must be effected between successive analyses.